Project Summary Effective vaccines remain elusive for many deadly diseases; therefore, it is critical that we better understand how the immune system generates a robust, neutralizing antibody response to vaccination so that we may enhance this type of response in future vaccine design. B cells are the producers of high-affinity antibodies, however, it is the CD4+ T cells that provide the cytokines and co-stimulatory molecules necessary to drive this B cell fate and establish long-term humoral immunity. This is why it is critical that we better understand the development and function of specific CD4+ T cell subsets involved in generating this type of response. After a primary infection or vaccination, some activated CD4+ T cells become a specialized subset specifically known to provide direct B cell help: T follicular helper (Tfh) cells. What commits T cells to the Tfh cell fate is still unknown. Furthermore, how the two main subsets of memory CD4+ T cells, Tcm and Tem, provide B cell help during a secondary challenge has never been directly compared due to a lack of experimental models that would allow for these types of studies. Remarkably, preliminary studies have revealed new findings regarding our previously characterized LLO TCRtg system that will allow me to uniquely address both of these gaps in understanding of adaptive immunity. Our two distinct LLO TCRtg CD4+ T cells recognize the same LLO epitope at the same affinity; however, they differ in their affinity for self-pMHC. The Hi5 cells have a higher affinity for self-pMHC (CD5hi) than the Lo5 cells (CD5lo). During a primary response, Lo5 cells develop into Tfh cells, while Hi5 cells generate essentially no Tfh cells. Since the LLO T cells recognize the same antigenic peptide at the same affinity, cell intrinsic differences are responsible for these functional variances. Furthermore, after contraction, Lo5 cells take on a Tem phenotype, while the Hi5 cells are Tcm cells. We hypothesize that tonic signaling is controlling the Tfh and memory cell fate decisions observed in our LLO model, and so we propose to: (1) directly compare ability to affect B cell outcome for two distinct CD4+ T cell memory populations: Tcm (Hi5) vs. Tem (Lo5), (2) elucidate aspects of tonic signaling previously uncharacterized, and (3) genetically manipulate tonic signaling in CD4+ T cells to determine the role it plays in Tfh differentiation and memory cell development and function. These findings will deepen our understanding of immunological memory and Tfh cell development and may reveal therapeutic targets for vaccine design and autoimmunity.